The transcriptional machinery of HIV-1 remains a therapeutic target that has not been exploited in current therapeutic regimens. The viral transactivator Tat and its cellular interaction partners play a critical role in the regulation of HIV transcription. We have obtained evidence that Tat also interacts with the p90 ribosomal S6 kinase 2 (RSK2). Dominant-negative RSK2 or siRNAs directed against RSK2 inhibited Tat transactivation, indicating that RSK2 is important for Tat function. Reconstitution of RSK2 in cells from individuals with a genetic defect in RSK2 expression (Coffin-Lowry syndrome) enhanced Tat transactivation. RSK2 is normally activated through the pp44/42MAPK/ERK signal transduction pathway after mitogenic stimulation and has been implicated in the phosphorylation of histone H3 and CREB/ATF transcription factors. We find that Tat activated the RSK2 kinase activity in cells. We propose to identify the mechanism of Tat-induced RSK2 activation by performing in vitro kinase assays of recombinant RSK2 in the presence of synthetic Tat. In addition, we will identify the target(s) of activated RSK2 at the HIV promoter by performing chromatin immunoprecipitation experiments using modification-specific antibodies against histone H3 and CREB/ATF transcription factors. We have recently obtained the first selective cell-permeable RSK inhibitor generated by the laboratory of Jack Taunton at the University of California San Francisco. Our preliminary experiments show that this inhibitor suppresses Tat transactivation. We will therefore study the relevance of RSK2 activation for HIV infection by testing the effect of RSK2 inhibition and RSK2 knockdown in T cells infected with HIV-1. We anticipate that these studies will substantially further our understanding of the role of RSK2 in HIV transcription and explore its potential as a novel target for anti-HIV therapy.